Fetal alcohol spectrum disorder (FASD) is the most common preventable cause of mental retardation in the Western World. Our research addresses FASD and has three broad goals: to better understand how ethanol disrupts fetal development; to identify biological factors that increase the susceptibility of women who drink during pregnancy to bear children with FASD; and to provide a scientific foundation for rational drug design to prevent or mitigate FASD. Our major focus is the L1 cell adhesion molecule, a developmentally critical protein that is implicated in the pathophysiology of FASD. Ethanol potently inhibits L1 adhesion and L1-mediated neurite outgrowth, and photolabeling identifies a binding pocket on the extracellular domain of L1 (L1-ECD) in which point mutations alter the effects of ethanol. The proposed research will test three principal hypotheses: 1) there is a specific location at the interface between L1 Ig1 and Ig4 at which ethanol alters L1 function by disrupting the horseshoe conformation of L1 that favors homophilic binding; 2) phosphorylation within the L1 cytoplasmic domain (CD) modulates L1 sensitivity to ethanol by causing conformational changes in the alcohol binding pocket of the L1 extracellular domain (ECD); 3) ethanol inhibition of L1 adhesion contributes to ethanol inhibition of L1-mediated neurite outgrowth (L1MNO). We will determine the effects of mutations that alter the ethanol binding pocket on the structure and function of L1. Structural changes will be detected by spinlabeling and double electron-electron resonance (DEER) protocols and by changes in the availability of sites for photolabeling by azialcohols. We will correlate structure with function by examining the effects of mutations on L1 adhesion, alcohol inhibition of L1 adhesion, and antagonist blockade of ethanol inhibition of cell adhesion using intact NIH/3T3 cells transiently transfected with L1 constructs. We will employ kinase inhibitors and mutations of specific kinase substrates in the L1-CD to determine how these sites regulate ethanol sensitivity. We will evaluate the effects of L1-CD phosphorylation on L1-ECD structure by determining whether L1-CD mutations prevent the formation of disulfide bonds between two cysteine reporters inserted within the alcohol binding pocket in the L1-ECD. We will also study masking or unmasking of antibody epitopes within the L1- ECD and alcohol inhibition of L1 adhesion in response to mutation of kinase substrates in the L1-CD. We will determine whether the pharmacology is similar for alcohol inhibition of L1 adhesion and L1MNO in cerebellar granule neurons. We will also study whether mutations that block ethanol inhibition of L1 adhesion also block ethanol inhibition of L1MNO. These experiments will expand our knowledge of how ethanol disrupts fetal development, why individuals differ in sensitivity to ethanol teratogenesis, and how drugs might be designed to reduce ethanol toxicity.